Since then, sporadic reports on findings of autoantibodies in this disease have been described, such as antibodies to retinal antigens, heat shock protein HSP of some strains of Streptoc
Trang 1Open Access
R1133
Vol 7 No 5
Research article
Identification of kinectin as a novel Behçet's disease autoantigen
Yu Lu1,2, Ping Ye1,2, Shun-le Chen2, Eng M Tan1 and Edward KL Chan3
1 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA
2 Department of Rheumatology, Shanghai Ren Ji Hospital affiliated to Shanghai Second Medical University, Shanghai, China
3 Department of Oral Biology, University of Florida, Gainesville, FL, USA
Corresponding authors: Yu Lu, luyu100@sina.com Edward KL Chan, echan@ufl.edu
Received: 14 May 2005 Revisions requested: 15 Jun 2005 Revisions received: 23 Jun 2005 Accepted: 4 Jul 2005 Published: 27 Jul 2005
Arthritis Research & Therapy 2005, 7:R1133-R1139 (DOI 10.1186/ar1798)
This article is online at: http://arthritis-research.com/content/7/5/R1133
© 2005 Lu et al.; licensee BioMed Central Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/
2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
There has been some evidence that Behçet's disease (BD) has
a significant autoimmune component but the molecular identity
of putative autoantigens has not been well characterized In the
initial analysis of the autoantibody profile in 39 Chinese BD
patients, autoantibodies to cellular proteins were uncovered in
23% as determined by immunoblotting We have now identified
one of the major autoantibody specificities using expression
cloning Serum from a BD patient was used as a probe to
immunoscreen a λZAP expression cDNA library Candidate
autoantigen cDNAs were characterized by direct nucleotide
sequencing and their expressed products were examined for
reactivity to the entire panel of BD sera using
immunoprecipitation Reactivity was also examined with normal control sera and disease control sera from patients with lupus and Sjögren's syndrome Six independent candidate clones were isolated from the cDNA library screen and were identified
as overlapping partial human kinectin cDNAs The finding that kinectin was an autoantigen was verified in 9 out of 39 (23%)
BD patient sera by immunoprecipitation of the in vitro translation
products Sera from controls showed no reactivity The significance of kinectin as a participant in autoimmune pathogenesis in BD and the potential use of autoantibody to kinectin in serodiagnostics are discussed
Introduction
Behçet's disease (BD) is a systemic vasculitic disease typified
by a triad of symptoms including recurrent oral ulcers, genital
ulcers and uveitis In addition, skin, joint, large vessels, nervous
system and gastrointestinal systems may be involved BD is a
global disease but has the highest prevalence in the region
along the ancient 'Silk Road' in China The etiopathogenesis of
the disease remains unclear but microbial agent triggers,
envi-ronmental factors, genetic predisposition, neutrophil
hyper-function, endothelial cell dysfunction and immunological
abnormalities involving both T and B cells have been
impli-cated Increasing amounts of research evidence supports the
possibility that it is an immune-mediated vasculitis, and that
abnormal T-cell and B-cell reactions and autoantigen-driven
autoimmunity play pivotal roles [1] Systemic lupus
erythema-tosus (SLE) is the prototypic systemic autoimmune rheumatic
disease with autoantibodies against cellular (particularly
nuclear) antigens, some of which are critically implicated in the
autoimmune pathology while others provide valuable serodiag-nostic markers for the disease Unlike the picture in SLE and other related rheumatic diseases, in BD, antinuclear antibod-ies and antibodantibod-ies to neutrophil cytoplasmic antigens etc are not present To date, since neither a specific autoantibody nor pathognomonic pathological index is available to help estab-lish the diagnosis of BD, it is largely or solely based on clinical manifestations [2], and a dilemma in diagnosis is not a rare occurrence in clinical practice Nevertheless, since the 1960s, there have been reports of autoantibodies against certain unknown components of human oral mucosa in sera of patients with BD Since then, sporadic reports on findings of autoantibodies in this disease have been described, such as antibodies to retinal antigen(s), heat shock protein (HSP) of
some strains of Streptococcus sanguis cross-reactive with
human HSP polypeptide [3], antibodies to endothelial cell anti-gens (AECA) and antibodies to α-tropomyosin [4,5], attesting
to the complicated humoral immune disorders in this disease
AECA = antibody to endothelial cell antigen; BD = Behçet's disease; DMEM = Dulbecco's modified Eagle's medium; HCC = hepatocellular
carci-noma; HSP = heat shock protein; IIF = indirect immunofluorescence; PBS = phosphate buffered saline; SjS = Sjögren's syndrome; SLE = systemic
lupus erythematosus.
Trang 2This investigation was aimed at defining target cellular
autoan-tigens using time-tested and well-established molecular
tech-niques Immunoscreening of expression libraries using BD
sera was used since this approach has been successfully
employed in the characterization of many clinically relevant
antigens in systemic rheumatic diseases such as SS-A/Ro
[6-9] and SS-B/La [10] antigens in Sjögren's syndrome (SjS)
and centromere antigen CENP-B [11] in scleroderma In
addi-tion, we have been successful in using this strategy to identify
interesting autoantigens that have other biological
signifi-cance Examples of these include NOR90/hUBF [12],
p80-coilin [13], Golgi autoantigens [14-16] and, more recently,
GW182 [17]
Materials and methods
Patients and sera
The currently used empirical criteria for the diagnosis of BD in
this study were the criteria proposed by the International Study
Group for BD (abbreviated as 'International Criteria') [2] The
study subjects of 39 Chinese BD patients comprised 17
males and 22 females, mean age 37 ± 11.3 years old, who
were divided into two subgroups: 25 typical BD patients
(Group I, satisfying the International Criteria) and 14 clinically
diagnosed BD patients who had recurrent oral ulcers and one
of the symptoms of genital ulcers, eye symptoms or skin
lesions as defined by the International Criteria, as well as
addi-tional symptom(s) closely related to BD as listed in the
Interna-tional Criteria, that is, gastrointestinal ulcerations, deep vein
thrombosis or arthralgia/arthritis without evidence that the
lat-ter symptoms might be related to any other disease (Group II,
defined as 'probable BD' in this study) Disease controls
included 10 patients with SLE and 10 with SjS, all satisfying
corresponding international classification criteria All BD
patients and disease controls involved in the study were
patients treated at the Rheumatology Department of Ren Ji
Hospital, Shanghai, China, where their clinical data and serum
samples were collected Twenty normal control sera were
ran-domly selected from healthy blood donors working in the same
hospital This study was approved by the institution review
board of Ren Ji Hospital which is affiliated with Shanghai
Sec-ond Medical University, and each patient involved gave
informed consent All serum samples were preserved at -20°C
or -70°C until use
Cell lines and cell extracts
HeLa (ATCC CCL 2.2) and T24 (human transitional cell
blad-der carcinoma) were obtained from the American Type Culture
Collection (Rockville, MD, USA) A bovine aortic endothelial
cell line was kindly provided by Dr Eugene G Levin from the
Scripps Research Institute (La Jolla, CA, USA) Cells were
cul-tured in DMEM containing 10% calf serum, harvested and
extracted in Buffer A (150 mM NaCl, 10 mM Tris-HCl, pH7.2,
0.5% Nonidet P-40) with protease inhibitor (Complete™;
Boe-hringer Mannheim, Indianapolis, IN, USA) For the preparation
of whole cell extract, 10 volumes of Laemmli gel sample buffer
[18] were added to the cell pellet, boiled for 3 min and stored
at -20°C until use
Western blot
Whole cell lysates from bovine aortic endothelial cell, HeLa and T24 cells were resolved individually by discontinuous 7.5% gel SDS-PAGE according to Laemmli's method [18]
Immunoblotting was performed as described by Towbin et al.
[19] with modifications Nitrocellulose strips were blocked with 3% nonfat milk in PBS containing 0.05% Tween-20 (PBS-T) and then incubated with BD patient sera and normal control sera (1:100 dilution) at room temperature for 1 h Fil-ters were washed extensively with PBS-T to remove any unbound antibodies Bound antibodies were detected with polyvalent, peroxidase-conjugated goat anti-human Ig and vis-ualized by incubating the nitrocellulose strips in chemilumines-cent reagents (NEN Life Science Products Inc., Boston, MA, USA) and exposing to Kodak XAR-5 films
Screening of phage cDNA expression library with antibody probes
Serum from a BD patient showing the highest antibody titer in immunoblotting was selected as a probe and used at a dilution
of 1:300 for initial immunoscreening of approximately 106
recombinants from a T24 cDNA expression library The latter was constructed in λZAPExpress vector (Stratagene, La Jolla,
CA, USA) and screened as previously described [20-22] All screenings were performed on duplicate isopropyl β -D-thioga-lactoside (IPTG) pre-impregnated nitrocellulose filters, and immunoreactive clones were detected by chemiluminescence Positive phages were subsequently plaque purified to 100%
by two repeated rounds of screening at low plaque densities Before screening the cDNA library, the BD serum was exten-sively adsorbed against bacteria and wild-type λZAP phage mixture to reduce background binding
Analysis of candidate cDNAs
Purified candidate plaques were subcloned in vivo into
pBK-CMV plasmids using ExAssist™ helper phage as recom-mended in the manufacturer's instructions (Stratagene) The recombinant pBK-CMV plasmids were then purified using QIAprep Spin Minprep Kit (Qiagen, Valencia, CA, USA) Restriction enzyme digestion of plasmids with EcoRI and XhoI and electrophoresis in a standard 1.0% agarose gel was used
to analyze the length of cDNA insert of each candidate plas-mid The complete nucleotide sequence was determined using Bigdye terminator sequencing and a semi-automated sequencer model 377 (ABI, Foster City, CA, USA) Both nucleotide and deduced amino acid sequences were analyzed for similarity with known sequences using BLAST search [23] and ExPASy Proteomics tools http://www.expasy.ch/www/ tools.html Secondary structure analysis for coiled-coil motifs was conducted with the software program COILS [24]
Trang 3Immunoprecipitation of in vitro translation products
Candidate cDNA clones were used as templates for in vitro
transcription and translation and the products were used as
substrates for immunoprecipitation to confirm the specificity of
reaction with BD sera In brief, 1 µg of the pBK-CMV plasmid
identified in the screening outlined above was added as
tem-plate in a 50-µl reaction for the coupled in vitro transcription
and translation reaction with a rabbit reticulocyte lysate system
(Promega, Madison, WI, USA) in the presence of 35
S-methio-nine (Trans-35S label; ICN Biochemicals, Costa Mesa, CA,
USA) and RNasin® Ribonuclease Inhibitor (Stratagene) as
recommended by the manufacturer (Promega) Translation
was carried out at 30°C for 1.5 h Products were analyzed in
a 12.5% gel SDS-PAGE and stored at -80°C for further
immu-noprecipitation analysis The in vitro translation proteins were
examined for reactivity by sera using immunoprecipitation
described [8,25]
Results and discussion
Autoantibody detection in sera from BD patients
Initial examination of a group of 39 BD patients using indirect immunofluorescence (IIF) on a HEp-2 cell substrate did not yield any characteristic nuclear or cytoplasmic staining pat-terns BD is thought by some to be a vasculitic disease involv-ing pathophysiology of endothelial cells, and antibody to endothelial cell antigen (AECA) has been reported Reports
on the prevalence of AECA have varied largely and alpha-eno-lase was reported as one of the putative target antigens [26]
In this study, the use of bovine aortic endothelial cells as sub-strate for IIF did not provide any additional data However, Western blot analysis of the BD sera began to show some interesting autoreactivity using cell lysates from both HeLa and bovine aortic endothelial cells HeLa cells were initially used for this analysis because they are commonly used in the labo-ratory as Western blot substrate Fig 1 illustrates the common reactivity to 49 kDa and 120 kDa proteins in the endothelial cell lysates These antigens were also detected in HeLa and T24 cells; the latter cell line was analyzed because our labora-tory at The Scripps Research Institute has produced an excel-lent expression cDNA library from the T24 line and the positive result with the T24 cell extracts allowed us to screen the T24 library Ig isotype analysis showed that all reactivity was largely IgG antibodies Since the 49 kDa and 120 kDa bands were observed in cell extracts from bovine as well as human cell lines, these autoantigens might be evolutionarily conserved
In total, nine out of 39 BD sera (23%) had autoantibody to the
49 kDa antigen and eight (20%) to the 120 kDa antigen Four
BD sera (10%) reacted with both proteins Additionally, sera that showed common reactivity to the 120 kDa protein also demonstrated a common band that migrated at ~150 kDa, although it appeared weaker than the 120 kDa band These antigens appeared to have different molecular weights than those of the known autoantigens in systemic rheumatic dis-eases In addition, other reactive bands were detected but they were not as commonly shared as the 49 kDa and 120 kDa bands The 49 kDa protein was shown to be distinct from 48 kDa SS-B/La or 50 kDa Jo-1 proteins (Fig 1) The 120 kDa antigen was also shown to migrate differently from alanyl tRNA synthetase in another Western blot analysis (data not shown) and did not share any apparent crossreactive epitopes with the 49 kDa antigen Western blot analyses of 20 normal con-trol sera did not show the reactivities observed with BD sera
In order to further characterize these autoreactivities, a serum sample from the Group I definitive BD patients with the strong-est reactivity to 49 kDa and 120 kDa antigens (Fig 1, lane 2) was selected as antibody probe for expression library screening
Kinectin identified as a novel BD autoantigen
After screening 500,000 clones from the T24 cell λ ZAPEx-press exZAPEx-pression library, seven immunoreactive clones were isolated and plaque purified in two to three rounds to achieve
Figure 1
Western blot analysis of autoantibodies in selected BD sera
Western blot analysis of autoantibodies in selected BD sera Whole
cell extracts from bovine aortic endothelial cells were separated by a
7.5% gel SDS-PAGE, transferred to nitrocellulose and probed with
serum dilutions of 1:100 Several sera showed reactivity to proteins of
49, 120 and 150 kDa The serum in lane 2 with the strongest reactivity
to the 49 kDa and 120 kDa bands was selected as the probe for the
expression library screening Molecular mass markers are shown on the
left NHS represents the normal control BD, Behçet's disease.
Trang 4100% homogeneity The cDNA inserts were subcloned in vivo
into pBK-CMV plasmids, analyzed by restriction digestion
using EcoRI and XhoI enzymes, and submitted to direct
nucle-otide sequencing across the polylinker arms The cDNA
inserts represented six independent clones designated BD41
(identical to BD44), BD481, BD42, BD47, BD482 and BD49
Their identities were established as overlapping partial cDNAs
of human kinectin, ranging from 1.9 kb to 3 kb (Fig 2a) The
full-length human kinectin (GenBank accession number
NM_182926[27]) has 4,816 bases containing an open
read-ing frame codread-ing 1,357 amino acid residues with molecular
mass 156 kDa All six cDNAs lacked the 5' portion of the
kinectin sequence to different degrees but spanned a
sequence of kinectin that extended to the 3'-untranslated
region Secondary structure analysis of kinectin protein using
the program COILS identified a long region of α-helical
coiled-coil domain that extended from amino acid residue 327 to the
C-terminus (Fig 2a, hatched boxes) In vitro coupled
tran-scription and translation of BD44 and BD42 clones directed
the synthesis of [35S]-methionine-labeled polypeptides that
migrated at 95 and 60 kDa, respectively, in addition to smaller
polypeptides (Fig 2b) These products had predicted
molec-ular weights of 103 kDa and 75 kDa
Kinectin was initially identified in chick embryo brain
micro-some as an integral membrane protein anchored in
endoplas-mic reticulum and involved in kinesin-driven vesicle motility
along microtubules [28,29] Kinectin consists of a 120-kDa
and a 160-kDa polypeptide interacting through the α-helical
coiled-coil domain to form a heterodimer [30] The full-length
kinectin is the 160 kDa polypeptide containing an N-terminal
transmembrane helix followed by a bipartite nuclear
localiza-tion sequence and two C-terminal leucine zipper motifs We
presume that the 120 kDa polypeptide detected in Western blot (Fig 1) is the truncated version of the 160-kDa polypep-tide, lacking the terminal first 232 amino acids [30] The N-terminus of the 160-kDa polypeptide consists of a transmem-brane domain that anchors kinectin to endoplasmic reticulum [30,31] This 120 kDa polypeptide is probably the predomi-nant form detected in the Western blot analysis (Fig 1) because of its preferential solubility due to the omission of the N-terminal transmembrane domain
Other functions for kinectin have been reported Yeast two-hybrid screen studies from several laboratories have revealed the interaction of the Rho family of GTPase with kinectin, and have shown the functional links among RhoG, kinectin and kinesin, with kinectin as a key effector of RhoG microtubule-dependent cellular activity [32] Kinectin was also identified as
an important constituent of integrin-based adhesion com-plexes, which link integrins to the cytoskeleton and recruit sig-naling molecules [33] A new study reported that a kinectin isoform lacking a major portion of the kinesin-binding domain
is very probably the most conservative form of kinectin; it does not bind kinesin but act as a membrane anchor for the transla-tion elongatransla-tion factor-1 delta in the endoplasmic reticulum [34]
Prevalence and specificity of anti-kinectin autoantibodies
The in vitro [35S]-methionine-labeled translation product of BD44, representing the largest recombinant kinectin fragment available, was used as the antigen substrate in an immunopre-cipitation assay Out of 39 BD patient sera, nine (23%) recog-nized the BD44 translation product (Fig 3), whereas sera from
20 normal controls, 10 SLE and 10 SjS patients did not show
Figure 2
cDNA clones obtained from expression library screen using a BD serum
cDNA clones obtained from expression library screen using a BD serum (a) Schematic diagram of the six overlapping cDNAs aligned with
pub-lished human full-length kinectin sequence (4,816 bp, GenBank accession number NM_182926, shown on top) The open box represents the cod-ing region of the full-length kinectin protein of ~156 kDa cDNA inserts from the six independent clones BD41/BD44, BD481, BD42, BD47, BD482 and BD49 represent N-terminal truncations and are predicted as coiled-coil domains (hatched) Ќ represents short sequences derived from
alterna-tive mRNA splicing in the 3'-untranslated region (b) Autoradiography of in vitro transcription and translation products of the candidate clones BD44
and BD42 labeled by [ 35 S]-methionine and analyzed on a 12.5% gel SDS-PAGE Products of BD44 and BD42 gave major bands with the highest molecular mass of 95 kDa and 60 kDa, respectively The lane marked as p90 represents an unrelated autoantigen used as a positive control for the
in vitro translation reaction Molecular markers are shown on the left BD, Behçet's disease.
Trang 5reactivity Among the nine anti-kinectin positive patients, six (6/
25, 24%) were from Group I (definitive BD) including the BD
patient whose serum was used in the immunoscreening of
expression cDNA library, and three (3/14, 21.4%) patients
were from the Group II (probable BD) in this study According
to the Fisher Exact Probability calculation (P = 1.00), there is
no statistically significant difference for antibody to kinectin
between the two groups The combined data substantiated
the finding that kinectin is an autoantigen that can be
recog-nized by sera from 23% of Chinese BD patients in this study
with at least one immunoreactive region or autoepitope
resid-ing within the BD44 encoded polypeptide
Currently, there are more than six diagnostic/classification
cri-teria for BD, among which the International Cricri-teria have been
applied most extensively due to its relatively high sensitivity
(91%) and specificity (96%) [2] As discussed above,
differen-tial diagnosis of BD might be confusing in clinical practice
since no specific laboratory test is available, and some
patients may have symptoms and signs strongly suggestive of
BD but do not fully satisfy the International Criteria, as in the
Group II (probable BD) patients in our study group A number
of investigators have pointed out that a comprehensive
analy-sis of the clinical data for a given patient is very important for
correct clinical diagnosis of BD, and that
classification/diag-nosis criteria, including the International Criteria, should be
fol-lowed but should not be exclusive The observation that three
out of 14 patients in the probable BD group also had antibody
to kinectin and the similar percentage of positive reactors
between this group and Group I (21.4% versus 24%)
sup-ports this notion The further use of non-clinical parameters
such as immunological biomarkers as adjuncts to identify BD patients could be of help in the classification of this disease entity
While our work was ongoing, anti-kinectin antibodies were reported in sera from patients with hepatocellular carcinoma (HCC) [35,36] and aplastic anemia [37,38] The first HCC report [35] identified kinectin as a tumor-associated antigen from the screening of an autologous cDNA library constructed from the cancer of a 30-year-old patient from Guangxi, China
This report stated that four out of five HCC patients tested were positive for anti-kinectin antibody [35] In 2004, another laboratory also reported the cloning of kinectin as a tumor-associated antigen from a (presumably) different 30-year-old Chinese HCC patient [36] In contrast, anti-kinectin antibodies were not detected in other studies of HCC patients associated with our laboratory [39,40] The reports of anti-kinectin antibodies in aplastic anemia are also very interesting
[37,38] The initial report by Hirano et al identified kinectin by
screening an aplastic anemia patient for candidate antigens using a Clontech human fetal liver cDNA expression library and it was concluded that seven out of 18 aplastic anemia patients were positive for anti-kinectin while none of the nor-mal or disease controls had this antibody [37] In their recent
report, Hirano et al reported that anti-kinectin antibodies were
found in 39% of aplastic anemia patients from the United States but only in three out of 30 (10%) cases in Japan [38]
In our study reported here, kinectin antibodies were only detected in BD patients and not in normal controls and SLE and SjS disease controls None of the BD patients with anti-kinectin had signs of HCC or aplastic anemia at the time of
Figure 3
Immunoprecipitation analysis of the 39 BD patients for autoantibody to kinectin
Immunoprecipitation analysis of the 39 BD patients for autoantibody to kinectin In vitro translation products of kinectin cDNA BD44 template was
used as the substrate Nine out of the 39 BD patient sera (23%, lanes 1–39) immunoprecipitated the BD44 translation products Molecular weight
standards are shown on the left The lane marked 'Total' shows the labeled translation product alone for comparison Lanes NHS1, NHS2, NHS3,
NHS4 are the four normal controls BD, Behçet's disease.
Trang 6diagnosis and at up to 4 years of follow-up Mapping of
epitope(s) recognized by anti-kinectin antibodies may shed
light on the question of whether different autoepitopes reside
within the kinectin molecule recognized by sera from different
diseases
Kinectin – a new member of coiled-coil cytoplasmic
autoantigens
We have recently reviewed the literature on the growing
number of cytoplasmic autoantigens rich in α-helical
coiled-coil domains as typified from our study of Golgi autoantigens
[41] Golgi autoantigens are generally high molecular weight
proteins between 100 and 350 kDa and rich in coiled-coil
domains in the central region with non-coiled-coil or globular
domains at both N and C termini Golgi autoantigens are
dis-played on the cytoplasmic face of the Golgi complex and are
not localized to apoptotic blebs during apoptosis [42]
Gian-tin, the highest molecular weight Golgi autoantigen reported,
is the predominant target of human Golgi complex
anti-bodies and multiple non-cross-reactive epitopes have been
mapped spanning the 350 kDa protein [43] Other high
molecular weight autoantigens with similar features have been
reported in cytoplasmic and mitotic organelles suggesting that
these selected proteins become autoimmunogenic based on
their subcellular association and molecular features [41] For
example, in the endosomal compartment, the two known
autoantigens are early endosomal protein EEA1 (180 kDa)
[44] and CLIP-170 (170 kDa) [45] There is also a series of
centrosomal autoantigens identified as coiled-coil-rich
pro-teins including pericentrin, a 220 kDa protein [46], ninein, a
protein with alternatively spliced products of 245 and 249 kDa
[47], Cep250 (250 kDa) and Cep110 (110 kDa) [48]
Centro-mere autoantigens have been described but the two
interest-ing ones related to this discussion are CENP-E [49] and
CENP-F [50]; both are high molecular weight proteins (312 to
400 kDa) and have the same type of overall structure as
dis-cussed above NuMA is another large coiled-coil protein
located at the mitotic spindle pole and is the most common
tar-get autoantigen in sera with mitotic spindle apparatus staining
[51] Non-muscle myosin (~200 kDa) is a cytoskeletal
autoan-tigen [52] that falls in the same group of high molecular weight
and coiled-coil-rich autoantigens These endosomal,
centro-somal, mitotic apparatus and intracellular autoantigens are,
like the golgins, proteins with high molecular weights and an
overall high content of coiled-coil domains The combination of
these two physical features in autoantigens may contribute to
the induction and production of autoimmune antibodies in
cer-tain disease states Kinectin is an integral membrane protein
largely confined to the endoplasmic reticulum [28,31] and it
fits into this new category of autoantigens that are large
coiled-coil rich proteins (≥100 kDa) in the cytoplasm
Conclusion
Here we report the detection of kinectin autoantibody in 23%
of Chinese patients with BD The identity of kinectin as a
BD-related autoantigen has not been reported to date Autoantibody reaction against kinectin in BD observed in this study further confirms the autoimmune involvement in BD and may provide new inroads into elucidating the immunopatho-genesis of the disease In an effort to clarify the association of
BD with antibody to kinectin, it is essential to measure anti-body to kinectin in larger patient populations including both
BD, probable BD and important autoimmune rheumatic dis-eases such as SLE, SjS, rheumatoid arthritis etc., as well as those diseases not easily differentiated from BD, such as recurrent aphthous oral ulcer, Reiter's syndrome, inflammatory bowel diseases etc On the other hand, further analysis of the association of anti-kinectin antibody with different manifesta-tions or disease 'subtypes' of BD is another important project Anti-kinectin is clearly only one of the antigen-antibody sys-tems identified because there were many other antibodies observed in the Western blot analysis of BD sera Using other sera for immunoscreening would probably lead to the identifi-cation of other potentially important antigen-antibody systems
Competing interests
The authors declare that they have no competing interests
Authors' contributions
YL performed the study and drafted the manuscript PY pro-vided technical help throughout the study SLC and EMT con-ceived the study, participated in the design and helped in the analysis of the data EKLC participated in the design of the study, interpreted data and helped to draft the manuscript All authors read and approved the final manuscript
Acknowledgements
This work was supported in part by National Institutes of Health Grants AI39645, AR42455, AI47859 (EKLC) and CA56956 (EMT), and National Nature Science Foundation of China Grant 30271225 (SLC).
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